Congenital malformations occur in up to 10% of babies born to diabetic women. Optimal glycemic control is difficult to achieve and maintain, and even transient exposure to hyperglycemia can cause malformations. This current proposal is formulated on the basis of our findings relative to the PKC pathway, which is critically involved i the induction of diabetic embryopathy. Because the molecular intermediates downstream PKC activation have not been determined, we have advanced a novel hypothesis that maternal hyperglycemia-induced PKCa/d activation reduces the expression of SIRT2 through DNA hypermethylation. SIRT2 downregulation prevents sustainable antioxidant enzyme expression and induces MARCKS hyperacetylation, which contributes to MARCKS phosphorylation. PKC-dependent SIRT2 downregulation and MARCKS phosphorylation in the developing neural tube are responsible for cellular stresses that cause neuroepithelial cell apoptosis and NTD formation. Aim 1 will determine whether PKCa and PKCd suppress SIRT2 expression through DNA hypermethylation in diabetic embryopathy. We hypothesize that that PKCa and PKCd increases the expression of de novo DNA methyltransferases (DNMT3A/B), which induce hypermethylation in the SIRT2 promoter and inhibit its expression. Aim 2 will determine the role of SIRT2 in PKC-induced cellular stress, MARCKS phosphorylation and NTD formation in diabetic embryopathy. Our working hypothesis is that downregulation of SIRT2 decreases antioxidant enzyme expression, which results in oxidative and endoplasmic reticulum stress, and leads to an increase in MARCKS acetylation and phosphorylation, which contributes to cellular stress. Aim 3 will determine the causal role of MARCKS phosphorylation in diabetes-induced cellular stress, apoptosis and NTD formation. We will test the hypothesis that PKCa/d-dependent MARCKS phosphorylation induces cellular stress and mitochondrial dysfunction, resulting in apoptosis and NTD formation. The success of this project will significantly fill the gaps in the current knowledge base regarding events that cause diabetic embryopathy.